Transmitting genetic information without creating deleterious genetic alterations is one of the cell?s most important tasks. Cells have evolved systems that check for and repair potentially lethal DNA damage. However, when these systems don?t work properly, DNA damage accumulates and causes genetic changes or cell death. Accumulation of genetic changes, which is defined as a genomic instability is frequently observed in various types of genetic disorders including cancers. Genomic instability has been documented as a preceding step for multiple inactivations of tumor suppressor genes and activations of proto-oncogenes. One type of genomic instability observed frequently in many cancers is gross chromosomal rearrangement (GCR). GCR includes translocations, deletions of chromosome arm, interstitial deletions, inversions, amplifications, chromosome end-to-end fusion and aneuploidy. Although little is known about the origin and mechanisms of GCRs observed in cancer cells, recent studies on genes mutated in inherited cancer predisposition syndromes have started to demonstrate that proteins that function in DNA damage responses, DNA repair, and DNA recombination, play crucial roles in the suppression of spontaneous and/or DNA damage-induced GCRs.The recent identification of strong correlations between genes responsible for genetic diseases including cancers and GCRs started to pinpoint the importance of GCRs. However, the mechanisms that are responsible for GCR formation were not studied in depth. One of the major reasons for this is that many genes that suppress and enhance GCR formation have not yet been discovered. [unreadable] [unreadable] 1) Suppression of GCR by yKu70-yKu80 heterodimer through DNA damage checkpoint[unreadable] The inactivation of either subunit of the Ku70-Ku80 heterodimer, which functions in nonhomologous end joining (NHEJ) and telomere maintenance, generates severe defects such as sensitivity to DNA damage, telomere shortening and increased gross chromosomal rearrangements (GCRs) that are frequently observed in many cancers. In order to understand the mechanism of Ku as a genome gatekeeper, we overexpressed the yKu70-yKu80 heterodimer and monitored the formation of GCRs. Ku overexpression suppressed formation of either spontaneously generated GCRs or those induced by treatments with different DNA damaging agents. Interestingly, this suppression was dependent on Ku?s interaction with DNA damage checkpoints, and not through NHEJ. We also demonstrate that the inactivation of telomerase inhibitor, Pif1 along with Ku overexpression or the overexpression of Pif1 in either yku70 or yku80 strains arrested cell cycle at the S phase in a DNA damage checkpoint dependent fashion. Lastly, Ku overexpression causes cell growth delay, which is dependent on intact Rad27. In summary, results presented here suggest that Ku functions as a genomic gatekeeper through its crosstalk with DNA damage checkpoints.[unreadable] 2) Regulation of Gross Chromosomal Rearrangements by Ubiquitin and SUMO Ligases.[unreadable] The Post-replication repair (PRR) bypasses DNA damage at the stalled replication folks. When replication machinery encounters a damaged DNA template, Rad6 and Rad18 monoubiquitinate Proliferating Cell Nuclear Antigen (PCNA) on lysine 164. Monoubiquitinated PCNA switches a replicative DNA polymerase to an error-prone translesion synthesis (TLS) DNA polymerase such as a DNA polymerase encoded by REV3/REV7 or an error-free TLS DNA polymerase eta encoded by RAD30. Accumulating evidence suggests a relationship between mutations in genes encoding PRR proteins and genomic instability. For instances, the targeted mutation of the mouse RAD18 gene in embryonic stem cells increased genomic instability including sister chromatid exchange, homologous recombination and illegitimate recombination. Mutations of XPV, the mammalian homolog of RAD30, that encodes TLS polymerase eta, were frequently found in xeroderma pigmentosum variant syndrome. Last year, we have shown that mutation of RAD5 or RAD18, ubiquitin ligases (E3) increases the de novo telomere addition type of GCR. The GCR suppression by Rad5 and Rad18 is dependent on the poly-ubiquitination of PCNA. In contrast, the SUMOylation of PCNA by Siz1 SUMO ligase at the same lysine residue is required for the generation of GCR. Inactivation of the homologous recombination pathway or the helicase, Srs2 reduced the elevated GCR rates by the rad5 or rad18 mutation. GCRs are therefore likely to be produced through the restrained recruitment of the HR repair pathway to the stalled replication forks. [unreadable] 3) Evidence sugestign that Pif1 helicase functions in DNA replication with the Dna2 helicase and DNA polymerase delta.[unreadable] The precise machineries required for two aspects of eukaryotic DNA replication, Okazaki fragment processing (OFP) and telomere maintenance, are poorly understood. In this work, we present evidence that Saccharomyces cerevisiae Pif1 helicase plays a wider role in DNA replication than previously appreciated and that it likely functions in conjunction with Dna2 helicase/nuclease as a component of the OFP machinery. In addition, we show that Dna2, which is known to associate with telomeres in a cell-cycle-specific manner, may be a new component of the telomere replication apparatus. Specifically, we show that deletion of PIF1 suppresses the lethality of a DNA2-null mutant. The pif1delta dna2delta strain remains methylmethane sulfonate sensitive and temperature sensitive; however, these phenotypes can be suppressed by further deletion of a subunit of pol delta, POL32. Deletion of PIF1 also suppresses the cold-sensitive lethality and hydroxyurea sensitivity of the pol32delta strain. Dna2 is thought to function by cleaving long flaps that arise during OFP due to excessive strand displacement by pol delta and/or by an as yet unidentified helicase. Thus, suppression of dna2delta can be rationalized if deletion of POL32 and/or PIF1 results in a reduction in long flaps that require Dna2 for processing. We further show that deletion of DNA2 suppresses the long-telomere phenotype and the high rate of formation of gross chromosomal rearrangements in pif1Delta mutants, suggesting a role for Dna2 in telomere elongation in the absence of Pif1.[unreadable] 4)Smc5-Smc6 mediate DNA double-strand-break repair by promoting sister-chromatid recombination.[unreadable] DNA double-strand breaks (DSB) can arise during DNA replication, or after exposure to DNA-damaging agents, and their correct repair is fundamental for cell survival and genomic stability. Here, we show that the Smc5-Smc6 complex is recruited to DSBs de novo to support their repair by homologous recombination between sister chromatids. In addition, we demonstrate that Smc5-Smc6 is necessary to suppress gross chromosomal rearrangements. Our findings show that the Smc5-Smc6 complex is essential for genome stability as it promotes repair of DSBs by error-free sister-chromatid recombination (SCR), thereby suppressing inappropriate non-sister recombination events.